Peculiarities of theophylline and prolactin interaction and calcium fluctuation from intracellular stores of porcine oocytes in the presence of estradiol

Using a fluorescent dye chlortetracycline, a study was made of the effect of estradiol on the interaction of theophylline and prolactin in the course of Ca2+ exit from intracellular stores of pig oocytes, isolated from ovaries at the stage of follicle growth. It is shown that in the presence of estradiol, prolactin does not stimulate Ca2+ exit from intracellular stores of pig oocytes. The action of theophylline similarly does not stimulate Ca2+ exit. Unlike, a joint effect of theophylline and prolactin on pig oocytes in the presence estradiol stimulated Ca2+ exit from intracellular stores of pig oocytes. These data demonstrated the influence of estradiol on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.     

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes was investigated using a fluorescent dye chlortetracycline. It is shown that in progesterone treated oocytes prolactin in concentration 50 ng/ml inhibits Ca2+ exit from intracellular stores of pig oocytes. Theophylline exerts the effect on prolactin Ca2+ exit from intracellular stores of pig oocytes. Employment of protein kinase C inhibitor cancelled inhibitory effect of prolactin and theophylline on Ca2+ exit from intracellular stores of pig oocytes. Ca2+ exit from intracellular stores of pig oocytes caused a joint influence of prolactin and GDP, and that of theophylline and GTP. The influence of protein kinase C inhibitor cancelled the stimulating effect of prolactin and GDP on Ca2+ exit from intracellular stores of pig oocytes also did not render any influence on the action of theophylline and GTP. These data suggest the influence of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.     

The influence of guanine nucleotides and protein kinase C on Ca2+ from intracellular stores of pigs oocytes stimulated by theophylline and cyclic AMP

Effect of guanine nucleotides and protein kinase C on Ca2+ exit from intracellular stores of pig oocytes, stimulated by theophylline and dbcAMP, was investigated using fluorescent dye chlortetracycline. Effect of cAMP on Ca2+ exit from intracellular stores of pig oocytes was not associated with activation of protein kinase C. In calcium-free medium, cAMP does not stimulate Ca2+ exit from intracellular stores of pig oocytes treated with GDP. In the presence of GDP, inhibition of protein kinase C activates Ca2+ exit from intracellular stores of pig oocytes on the action of cAMP. These data suggest the existence of different effects of guanine nucleotides on Ca2+ exit from intracellular stores of pig oocytes stimulated by cAMP.     

Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes

Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes was investigated using fluorescent dye chlortetracycline. It was shown that in the presence of estradiol neithert theophylline nor prolactin stimulated Ca2+ exit from intracellular stores of oocytes. Unlike, the common action oftheophylline and prolactin, also in the presence of estradiol, stimulated Ca2+ exit from intracellular stores. Inhibition of protein kinase C inhibits Ca2+ exit from intracellular stores in common action of theophylline and prolactin. These data suggest an obvious influence of estradiol on Ca2+ exit from intracellular stores of pig oocytes stimulated by theophylline and prolactin.     

Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.     

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of prolactin stimulated pig oocytes

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of pig oocytes activated by prolactin was investigated, using the fluorescent dye chlortetracycline. In the presence of extracellular calcium, the inhibitor of protein kinase C Ro 31-8220 increased calcium exit from intracellular stores in pig oocytes after prolactin treatment. In calcium-free medium, Ro 31-8220 exerted effect on calcium release from intracellular stores. In calcium-free medium, prolactin did not stimulate calcium release from intracellular stores of oocytes in the presence of thimerosal, while in the presence of protein kinase C inhibitor, prolactin increased Ca2+ content from intracellular stores in such oocytes. These data suggest a direct involvement of protein kinase C in the processes of regulation of Ca2+ exit from intracellular stores of pig oocytes stimulated by prolactin.     

Ca2+ release from intracellular stores of pig oocytes during different stages of growth

Ca2+ release from intracellular stores of pig oocytes was investigated using the Ca(2+)-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl clue (BCB) staining. The stained oocytes (BCB "+") were determined as the ones that completed their growth, while the stainless ones (BCB "-") were determined as those in the final stages of growth. In the BCB "+" and BCB "-" oocytes, prolactin, theophylline, GTP, and GDP cause Ca2+ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca2+, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2+. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca2+ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca2+ release from intracellular stores in growing and grown pig oocytes.     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.     

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.     

Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei.

During the successive interphases of cleaving mouse embryos the nuclear periphery diminishes its reactivity to anti-lamin A and C antibodies. This developmentally regulated characteristic can be modified by exposure of the blastomere nuclei to metaphase II (M II) oocyte cytoplasm followed by activation. In the current study we define the cytoplasmic conditions necessary for this modification of 8-cell and 16-cell stage nuclei in hybrids obtained by fusion with metaphase II arrested oocytes, oocytes at various time points after parthenogenetic activation, naturally fertilized eggs (zygotes) and interphase 2-cell embryo blastomeres. The intensity of fluorescence obtained with anti-lamins A/C in the blastomere nuclei increases as a result of fusion with freshly activated oocytes or early zygotes (first 3.0-5.5 h in the case of parthenogenetic activation), and not when eggs or 2-cell blastomeres advanced in interphase are used as partners for fusion. This transformation of the A/C lamin pattern is correlated with the ability to promote pronucleus-like growth of blastomere nuclei in hybrids. Blastomere nuclei introduced into M II-arrested oocytes undergo premature chromatin condensation and dissolution of the nuclear lamina. The results are discussed with regard to certain particularities of the first embryonic interphase of the mouse and the potential involvement of nuclear lamins in pronuclear growth.     

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.     

Effect of calcium ion on the maturation of cumulus-enclosed pig follicular oocytes isolated from medium-sized graafian follicles

Porcine follicular oocytes from medium-sized follicles (3-5 mm in diameter) were cultured in modified Hank's balanced salts solution (MHBS) to which pyruvate, lactate, and glucose were added as an energy source. Bovine serum albumin (0.4%) was added as a protein source and the oocytes were cultured for 42 h at 37 degrees C in 5% CO2 in air. In this medium porcine oocytes underwent 80-90% nuclear maturation after 42 h. Oocytes were cultured in MHBS with various amounts of CaCl2 as well as in the presence of verapamil, a Ca2+ channel blocker, and the divalent cationophore A23187. It was found that the lowest concentration of Ca2+ required for oocyte maturation was around 0.0265-0.053 mM. Such a requirement for Ca2+ in the culture medium extended through metaphase II. If Ca2+ was omitted during the final 4 h of culture, the metaphase II chromosomes appeared extremely condensed or degenerated. Verapamil at a level of 0.2 mM inhibited germinal vesicle breakdown or resulted in degeneration, whereas lower concentrations did not affect oocyte maturation. In the presence of 0.02 mM verapamil, the maturation of cumulus-enclosed oocytes was not affected, whereas at the same dose of verapamil the maturation of denuded oocytes was inhibited. Less than 3.8 X 10(-7) M divalent cationophore did not inhibit oocyte maturation. Maturation was inhibited by 3.8 X 10(-7) and 3.8 X 10(-6) M divalent cationophore. In conclusion, maintenance of oocytes in a nondegenerated state also requires the constant presence of Ca2+ in the culture medium.     

Identification of signal transduction pathway in fresh and vitrified porcine oocytes

Signal transduction pathway under the influence of somatotropin have been identified basis on the analysis of Ca2+ release from intracellular stores of fresh and vitrified porcine oocytes using inhibitory analysis. Somatotropin and GTP individually stimulated Ca2+ release from intracellular stores. The joint action of somatotropin and GTP activated additional Ca2+ release from intracellular stores both in fresh and vitrified porcine oocytes. Treatment of the oocytes with inhibitor of protein kinase C caused no additional Ca2+ release from intracellular stores. Ca2+ release from intracellular stores stimulated by GTP was connected with phosphate hydrolysis. Moving between intracellular Ca2+ depots stimulated by GTP was not determined by phosphate hydrolysis. Inhibitor of protein kinase C and microtubules were involved in the interaction of various intracellular depots. The data obtained suggest that signal transduction pathway in porcine oocytes do not change after vitrification.     

Effects of guanine nucleotides and protein kinase C on prolactin-stimulated release of Ca from intracellular stores of pig oocytes

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca2+ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca2+ and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca2+-free medium, prolactin did not stimulate Ca2+ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.     

Ca<Superscript>2+</Superscript> release from intracellular stores of pig oocytes at different stages of growth

Ca²⁺ release from intracellular stores of pig oocytes was investigated using the Ca²⁺-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl blue (BCB) staining. The stained oocytes (BCB “+”) were determined as the ones that completed their growth, while the stainless ones (BCB “−”) were determined as those in the final stages of growth. In the BCB “+” and BCB “−” oocytes, prolactin, theophylline, GTP, and GDP cause Ca²⁺ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca²⁺, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2⁺. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca²⁺ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca²⁺ release from intracellular stores in growing and grown pig oocytes.     

Fertilization and InsP3-induced Ca2+ release stimulate a persistent increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. We have investigated Ca2+ -induced cyclin destruction in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. We find no evidence for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. We conclude that increases in cytosolic Ca2+ induce transient changes in autofluorescence and that the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+ -induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction.     

Nuclear transplantation in early pig embryos

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.     

Characterization of calcium oscillation patterns in caprine oocytes induced by IVF or an activation technique used in nuclear transfer

Routine activation of nuclear transfer (NT) eggs involves the application of a single intracellular calcium [Ca2+]i rise, stimulated by an electrical pulse, as opposed to [Ca2+]i oscillations, which is the natural mode of sperm-induced activation at fertilization in all mammalian species tested to date. It has yet to be shown that caprine oocytes exhibit an increase in calcium at fertilization in a manner similar to other mammals. The objective of the present study was to evaluate and characterize the ([Ca2+]i) oscillation patterns of caprine metaphase II (MII) oocytes during IVF and during an activation techniques used in nuclear transfer. Additionally, the effect of cytochalasin B (cyto B) in the NT process was evaluated for its impact on [Ca2+]i oscillations and subsequent embryo development. Mature in vitro and in vivo derived caprine oocytes were activated by 5 microM ionomycin, an electrical pulse(s), or IVF. The intracellular Ca2+ response was determined using the [Ca2+]i indicator Fura-2 dextran (Fura-2D). Ova treated with ionomycin or stimulated by an electrical pulse exhibited a single [Ca2+]i rise, whereas IVF-derived oocytes showed oscillations. IVF [Ca2+]i showed some variation, with 62% of in vitro matured oocytes exhibiting oscillations, whereas 8% of in vivo matured oocytes exhibited oscillations demonstrating a correlation between [Ca2+]i responses and maturation technique. Knowing the [Ca2+]i profile of activated eggs, one may be able to optimize the activation methodology used in a production nuclear transfer setting which could potentially improve development to term for NT embryos.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.